No abstract

During periodontal regeneration, inhibition of gingival downgrowth is necessary to promote migration of mesenchymal cells into the defects. Transforming growth factor (TGF)-β is a pleiotropic cytokine that has numerous cell functions, including regulation of epithelial growth. Recent studies have shown that Smad2, a downstream transcription factor of TGF-β, plays crucial roles in wound healing in the epithelia. Therefore, we investigated the effects of Smad2 overexpression on re-epithelialization of gingival wounds. Transgenic mice overexpressing smad2 driven by the keratin 14 promoter (k14-smad2) were confirmed to have significant Smad2 phosphorylation in gingival basal epithelia. Punch wounds were made in the palatal gingiva, and wound healing was assessed histologically for 7 days. Re-epithelialization was significantly retarded on day 2, while collagen deposition was enhanced on day 7 in k14-smad2 compared with wild-type mice. Moreover, expression of keratin 16 (K16), an indicator of keratinocyte migration, was significantly inhibited in wound-edge keratinocytes in k14-smad2. The inhibition of K16 coincided with the induction of Smad2 in the corresponding epithelia, while BrdU incorporation was unaffected. These results indicated that Smad2 has inhibitory effects in regulating keratinocyte migration during gingival wound healing. TGF-β/Smad2 signaling mediating alteration of K16 expression must be tightly regulated during periodontal regeneration.

The signaling activation triggered by overexpression of Smad2 was dependent on TGF-β type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

The periodontal ligament (PDL) cells contain heterogeneous mesenchymal cell populations, which have the ability to differentiate into cells that produce adjacent mineralized tissues and abundant extracellular matrix (ECM). ECM is essential not only for the homeostasis of the periodontal tissue, but also for controlling the differentiation of the PDL cells. The process of differentiation involves mechanotransduction, which links the ECM to the cytoskeleton. The present study investigated the roles of Rho-associated coiled-coil containing protein kinase (ROCK) signaling, a crucial regulator of the cytoskeleton, during ECM-mediated osteogenic differentiation of PDL cells in vitro. The PDL cells were isolated from human periodontal ligaments of extracted teeth and cultured in osteogenic medium with or without Y-27632, a pharmacological inhibitor of ROCK. ECM-coated plates were used for ECM-mediated differentiation. The osteogenic phenotype was evaluated at different time points by real-time RT-PCR for the gene encoding alkaline phosphatase (ALP) and an ALP activity assay. The effects of ROCK on cytoskeletal changes and ECM synthesis were examined by immunofluorescence analysis. Y-27632 significantly inhibited ALP at the mRNA and protein activity levels in the late stage of differentiation; concomitantly, the actin filament content and the extracellular levels of collagen-I and fibronectin were markedly decreased by Y-27632. Exogenous collagen-I and fibronectin temporally increased ALP activity, with fibronectin showing a more pronounced effect. Importantly, ECM-mediated differentiation was almost completely inhibited by Y-27632. These findings indicated that ECM-mediated differentiation is dependent on ROCK signaling, and ROCK signaling contributes to the establishment of the ECM microenvironment for PDL cell differentiation.

No abstract